骨髓增生异常综合征的造血抑制活性

采用体外单层琼脂培养技术，观察了10例骨髓增生异常综合征(MDS)患者骨髓和外周血单个核细胞条件培养液和血清对正常骨髓粒单系祖细胞集落形成影响．并与16例急性髓细胞性白血病(AML)患者进行比较．结果表明，与AML-样，MDS患者骨髓、外周血单个核细胞条件培养液和血清可抑制粒单系祖细胞集落形成，存在白细胞和关抑制活性和血清抑制活性，推测这种抑制活性可能与患者粒细胞减少有关。     

Interaction of anti-thymocyte serum with haematopoietic stem cells: II. Stimulation of colony formation in vitro

When mouse haematopoietic cells are incubated in vitro in appropriate dilutions of horse anti-mouse thymocyte serum or globulin (HAMTS or HAMTG), the number of in vitro colonies formed by such cells is increased. There was a good correlation between the immunosuppressive potency and the colony-enhancing property of a given serum preparation. Correlation between the thymocytotoxicity titre and the colony enhancing property of the sera was poor. For colony enhancement, it was found necessary to have an added source of colony stimulating factor in the medium, whether in the form of mouse serum or human urine. Unlike the effect produced by exposure of bone marrow cells to certain antigens, HAMTG or HAMTS enhanced colonies in the absence of the alpha-globulin component of mouse serum. Thymus-derived immunocompetent cells are not involved in enhancement since this effect was also observed using foetal liver cells. It appears likely that the interaction of an antibody on the surface of some cell or cells involved in in vitro colony formation permits a larger fraction of the colony-forming cells to proliferate in vitro.     

Cross-reactivity studies of horse, goat and rabbit anti-lymphocyte globulin

In the sera of ten normal humans and twenty-eight candidates for organ transplantation, the passive haemagglutination test detected a 50% incidence of preformed antibodies of low titre directed against horse serum. Such antibodies were also found to cross react with goat or rabbit sera in most instances. Seventeen of the organ recipients were later studied after the institution of treatment with horse antihuman-lymphocyte globulin (ALG). The incidence of anti-horse-serum antibodies rose to 100%. At the same time, an increased activity against goat serum developed; cross-reactions against rabbit serum were also demonstrated but to a less pronounced degree. With immunoelectrophoresis and Ouchterlony diffusion tests, it was shown that the cross-reactivity was to similar although not necessarily identical protein components of the different heterologous sera.

These data suggest that there is an inherent risk in switching from one ALG to another, particularly if horse and goat derivatives are used sequentially. Since rabbit ALG does not cross react so extensively with horse or goat serum it would be predicted to be a relatively safer second-line agent.

These expectations seemed to have been realized in six patients who were given more than one kind of ALG, always beginning with horse globulin. When goat ALG was administered second, anaphylactic reactions tended to appear early, but when the rabbit product was given second or third, it was relatively well tolerated.

     

In vitro bone marrow cell migration to supernatants prepared from thymic epithelial cell cultures

Thymic epithelial cell cultures were established from neonatal CBA/J mice by inhibition of fibroblast overgrowth. Epithelial cells were identified by their cobblestone appearance in culture, by the presence of keratin, and by ultrastructural analysis demonstrating desmosomes. Supernatants were prepared by incubation of confluent cultures of these cells in serum-free media. Using blind well chambers, these supernatants were chemoattractive to murine bone marrow cells, enriched for immature lymphoid cells, and decreased the myeloid to lymphoid ratio in the migrating cell population. Interleukin 2 had no effect as a modulator of this chemoattraction, nor did it possess chemoattractive properties. Supernatants prepared from epidermal growth factor stimulated thymic epithelial cells possessed significantly enhanced chemoattractive properties to bone marrow cells, and was found to be a thymic epithelial cell mitogen. Supernatants from serum-free cultures of thymocytes also induced a significant migration of bone marrow cells but were found to enrich for mature myeloid cells.     

Cytology and histology of haemopoietic cell transplantation under the influence of antilymphocyte serum (ALS) in mice. I. The in vivo effect of antilymphocytic globulin on haemopoietic tissue

C57BL/6J mice were treated with ALS, ALG, normal rabbit serum (NRS) or IgG of the rabbit for a period of 6 days. To study the xenogeneic antigenicity of ALG and its antilymphocytic effect separately, half the mice were made tolerant to IgG of the rabbit. While a short schedule of injections of ultracentrifuged IgG induced tolerance to IgG, this tolerance was eliminated by the subsequent treatment with ALG. A prolonged injection schedule over 44 days did, however, lead to a tolerance which persisted after ALG treatment. [¹³¹I]IgG elimination proved to be a more sensitive control of tolerance than immunodiffusion.

Semiquantitative cytology revealed a decline in lymphocytes, erythroblasts and megakaryocytes in the bone marrow and erythroblastosis and megakaryocytosis in the spleen. Cells of the myeloid series invariably increased in number. The effect on erythroblasts persisted after removal of contaminating haemagglutinins. ALG-treated animals intolerant to IgG had about four times more splenic plasma cells than ALG-treated mice tolerant to IgG. The 6 days' treatment with ALS reduced the absolute number of nucleated bone marrow cells by 16% and doubled the spleen indices. Under this treatment, relative and absolute cell counts revealed an ALG-induced shift of erythroblasts, megakaryocytes and perhaps also of plasma cells from the bone marrow to the spleen. The effect of NRS or IgG was similar to that of ALS or ALG but remained on a lower level.

The remarkable changes in the histologic structure of the splenic follicles after ALG treatment were almost completely reversed by a prior induction of tolerance to IgG.

     

Differential induction of hematopoiesis and immune suppressor cells in the bone marrow versus in the spleen by Lewis lung carcinoma variants

Mice bearing large (greater than or equal to 3 g) metastatic and nonmetastatic Lewis lung carcinoma (LLC) tumors were studied to determine if the tumor variants differentially induced bone marrow versus splenic hematopoiesis and the appearance of hematopoiesis-associated immune suppressor cells. The metastatic LLC-C3 and nonmetastatic LLC-C8 tumors were equal in their stimulatory effects in vivo on both the number of bone marrow myeloid progenitor cells (CFU) and the appearance of bone marrow immune suppressor cells. In contrast, the tumor variants differed in their effects on the spleen, with the metastatic tumors causing a more pronounced increase in the number of nucleated cells and CFU, a reduced blastogenic responsiveness to concanavalin (Con-A), and an increased suppressor cell activity than nonmetastatic LLC-C8 tumors. The splenic suppressor cells of mice bearing large LLC-C3 tumors resembled the bone marrow suppressor cells which we previously described (Young et al.: Cancer Res. 47, 100, 1987) in that they were nonadherent to nylon wool, sensitive to treatment with L-leucine methyl ester, insensitive to treatment with complement and Thy-1.2, MG-1.2, asialo-GM1, or anti-IgM antibodies, and mediated their suppression through a mechanism which was only partially indomethacin sensitive. The stimulatory effects on hematopoiesis and suppressor cells by the LLC variant tumors may have been mediated by the tumor-derived colony stimulating factor (CSF) activities. Bone marrow cell proliferation and colony formation were stimulated in vitro by culture supernatants of metastatic LLC-C3 cells and, to a lesser degree, of nonmetastatic LLC-C8 cells. These colony-stimulating factor (CSF)-containing supernatants also induced normal bone marrow cells to become immune suppressive. In contrast, supernatants of only LLC-C3 cells, and not of LLC-C8 cells, stimulated in vitro growth of splenic CFU from LLC-C3-bearing mice; spleen cells from normal mice and from LLC-C8 bearers were unresponsive to supernatants of the LLC variants. These results suggest that CSF produced by either the metastatic LLC-C3 or the nonmetastatic LLC-C8 tumors could concurrently stimulate bone marrow hematopoiesis and the appearance of bone marrow suppressor cells. However, the metastatic LLC-C3 tumor cells, and not the nonmetastatic LLC-C8 cells, could also cause expansion of progenitor cells and hematopoiesis to the spleen and, consequently, induce the appearance in the spleen of hematopoiesis-associated immune suppressor cells.     

Thermal Injury Increases the Number of Eosinophil Progenitors in Rat Spleen and Bone Marrow

We have investigated the effects of thermal injury upon myelopoiesis. IL-3, GM-CSF, and IL-5 were used to stimulate myeloid colony formation. IL-3 induces early myeloid progenitors and a more developed myeloid progenitor, the granulocyte-macrophage colony-forming unit (GM-CFU), to multiply and develop into mature myeloid cells. GM-CSF induces GM-CFU to become mature myeloid cells, while IL-5 induces eosinophil progenitors to become mature eosinophils. Stem Cell Factor (SCF) + IL-6 and FLT3 ligand, which have no effect on colony formation by themselves, were used to enhance the effects of IL-3 and GM-CSF, respectively. We found that thermal injury increased the number of early myeloid progenitors and GM-CFU in the spleen with either IL-3 or GM-CSF as a stimulant. Thermal injury increased the number of early myeloid progenitors in the bone marrow when GM-CSF, but not IL-3, was used to stimulate colony growth. Also, thermal injury increased the numbers of eosinophil progenitors in rat spleen and bone marrow and increased splenic levels of IL-5 mRNA.     

The effect of a calf thymus acid lysate on bone marrow cell growth in vitro

Granulocyte-macrophage colony forming units (CFU-GM) were studied in cultures of bone marrow from 16 apparently healthy normal controls, 9 patients with the myelodysplastic syndrome, 5 patients with myeloproliferative disease and 2 with myeloma. Supernatants from non-stimulated 72 hr cultures of nonadherent mononuclear blood cells ("lymphocytes") stimulated the forming of an average of 38.4 colonies per 100,000 cells from normal marrow. The addition of GIBCO's commercial conditioned medium or of a medium produced by lymphocytes stimulated with different concentrations (5, 10 and 20 mcg/ml) of an acid lysate of thymus (thymomoduline), increased growth to 65.2 - 55.4 colonies (p less than 0.001 to 0.05). Similarly, a significant increase (p less than 0.05) was found in the number of clusters and colonies formed in cultures of marrow from patients with the myelodysplastic syndrome. In contrast, no growth was found when the thymus acid lysate was added directly to the bone marrow cultures, suggesting that the lysate induces the production of colony stimulating activity by lymphocytes, but does not contain it. Similarly no significant increase was found as regards the initially high number of colonies from the five patients with myeloproliferative disease, or as regards the initially low number in the two myeloma patients.     

Tenascin immunoreactivity in normal and pathological bone marrow.

AIMS: To determine the distribution of tenascin in normal and pathological bone marrow. METHODS: 48 different bone marrow lesions were studied immunohistochemically using a monoclonal antibody to tenascin. RESULTS: Tenascin immunoreactivity was found in lesions with increased fibrosis and high numbers of reticular fibres. The strongest immunoreactivity was found in myelofibrosis. Bone marrow from acute and chronic myeloid and lymphatic leukaemias showed weak or moderate immunoreactivity. In hyperplasias inconsistent reticular tenascin immunoreactivity was found; in normal bone marrow, only a few scattered positive fibres were occasionally seen. CONCLUSIONS: Tenascin was generally observed in conditions in which megakaryocytic hyperplasia was a feature. This is in line with the notion that tenascin synthesis in bone marrow fibroblasts is stimulated by TGF-beta which is synthesised by the megakaryocytic lineage. Tenascin also contains EGF-like repeats. It might therefore function as a growth promoter and in this way could also stimulate synthesis of other matrix components. On the other hand, tenascin could function as an adhesive molecule to some cells of the bone marrow. The presence of tenascin in many pathological states of the bone marrow suggests that it may have a role in their pathogenesis and that it also could be a potential marker of disease.     

Anti-horse globulin antibodies in human sera

Organ transplant patients who had received ALG, patients with rheumatoid arthritis and normal persons were studied for serum antibodies to horse globulins. Although normal individuals rarely show the presence of anti-horse antibody, rheumatoid patients with high titres of rheumatoid factor usually show anti-horse globulin agglutinins in their IgM globulins and these agglutinins are considered to be a manifestation of their `rheumatoid factors'. These agglutinins are readily absorbable with aggregated human γ-globulin. On the other hand, although most transplant patients administered ALG produce anti-horse antibody, it is usually produced as an IgG globulin and it is not absorbable with aggregated human γ-globulin.     

Murine embryonic stem cells secrete cytokines/growth modulators that enhance cell survival/anti-apoptosis and stimulate colony formation of murine hematopoietic progenitor cells

Stromal cell-derived factor (SDF)-1/CXCL12, released by murine embryonic stem (ES) cells, enhances survival, chemotaxis, and hematopoietic differentiation of murine ES cells. Conditioned medium (CM) from murine ES cells growing in the presence of leukemia inhibitory factor (LIF) was generated while the ES cells were in an undifferentiated Oct-4 expressing state. ES cell-CM enhanced survival of normal murine bone marrow myeloid progenitors (CFU-GM) subjected to delayed growth factor addition in vitro and decreased apoptosis of murine bone marrow c-kit(+)lin- cells. ES CM contained interleukin (IL)-1alpha, IL-10, IL-11, macrophage-colony stimulating factor (CSF), oncostatin M, stem cell factor, vascular endothelial growth factor, as well as a number of chemokines and other proteins, some of which are known to enhance survival/anti-apoptosis of progenitors. Irradiation of ES cells enhanced release of some proteins and decreased release of others. IL-6, FGF-9, and TNF-alpha, not detected prior to irradiation was found after ES cells were irradiated. ES cell CM also stimulated CFU-GM colony formation. Thus, undifferentiated murine ES cells growing in the presence of LIF produce/release a number of biologically active interleukins, CSFs, chemokines, and other growth modulatory proteins, results which may be of physiological and/or practical significance.     

Cellular responsiveness in vitro: the effect of mycobacterial glycopeptides, glycolipids and peptidoglycolipids on the in vitro growth of haemopoietic colony-forming cells.

Incorporation of glycopeptide, glycolipid and peptidoglycolipid preparations from the culture fluid or organisms of a variety of mycobacterial species in suboptimally stimulated cultures of normal mouse bone marrow cells increased the responsiveness of colony-forming cells to colony-stimulating factor, as measured by enhanced colony formation. Possible mechanisms of action and the significance of enhanced colony growth in relation to the mode of action of mycobacterial adjuvants are discussed.     

A colony assay system that detects B cell progenitors in fresh and cultured bone marrow

This report describes a colony assay system, based on methods used to grow myeloid precursors in semisolid medium, in which B cell progenitors can be grown. The formation of these B cell progenitor colonies is dependent upon soluble mediators from a stromal cell line known to support B lymphopoiesis. In initial experiments a double layer culture system was employed in which target cells in methylcellulose medium were separated from an adherent layer of S17 stromal cells by an agar interface. Target cells were harvested from Dexter type long-term bone marrow cultures at a time after transfer to the lymphoid Whitlock-Witte conditions, when myeloid progenitors were depleted and mature B cells had not yet appeared. On day 15 of culture a colony could be identified that contained several hundred tightly clustered lymphoid cells. There was a linear relationship between the number of cells plated and the number of colonies that developed. Identically appearing colonies were also observed in agar using fresh bone marrow cells as targets with either an underlayer of S17 cells or S17 conditioned medium to potentiate colony growth. Lymphoid colonies derived from fresh bone marrow appeared on days 6 and 14 of growth. A proportion of the cells from the fresh or cultured marrow derived colonies expressed the B220 antigen and cytoplasmic mu heavy chains, but surface IgM was never observed. Cell depletion experiments on antibody coated plates demonstrated the colony forming unit to be B220 antigen positive, surface IgM negative, and replating experiments indicated the colonies were lymphoid restricted in their differentiative potential.     

Regulation of murine hematopoiesis by arachidonic acid metabolites

Arachidonic acid metabolites have been shown to exert a variety of regulatory effects on cellular activation and proliferation. Recently, a role for these products as regulators of hematopoiesis was suggested and evidence provided that products of the lipoxygenase pathway, specifically leukotrienes, are essential for human myeloid colony formation in vitro. In this report the broader role of these metabolites in hematopoiesis was examined using murine bone marrow stem cell assays for both myeloid and lymphoid cell lines. The effects of lipoxygenase and/or cyclooxygenase pathway inhibitors on stem cell colony formation were evaluated and compared to qualitative and quantitative changes in arachidonic acid metabolism that occurred in similarly treated bone marrow cell cultures. Interruption of the lipoxygenase pathway by esculetin or nordihydroguaiaretic acid resulted in decreased colony formation in both lymphoid and myeloid stem cells. This inhibition of colony growth was partly reversed by the addition of leukotrienes and was particularly evident in B-cell progenitor cultures to which was added LTB4. Inhibition of the cyclooxygenase pathway by indomethacin or ibuprofen had a slight stimulatory effect on myeloid colony formation, while slightly inhibiting the formation of lymphoid colonies. These results support a direct role for lipoxygenase products in myeloid colony formation and lymphoid stem cell proliferation. A more complex role for cyclooxygenase metabolites in the hematopoietic process appears probable.     

Ligand-activated natural killer T lymphocytes promptly produce IL-3 and GM-CSF in vivo: relevance to peripheral myeloid recruitment

Natural killer (NK) T cells are prominent for their prompt IL-4 and IFN-gamma production upon TCR ligation that enables them to influence acquired immune responses. In the present study we provide evidence that the regulatory functions of this particular T cell subset extend to the myeloid compartment of bone marrow and spleen through its production of hematopoietic growth factors. Bone marrow and spleen NKT cells responded to a single injection of their specific ligand alpha-galactosylceramide (alpha-GalCer) by producing both IL-3 and granulocyte-macrophage colony stimulating factor (GM-CSF), whose colony-stimulating activity became detectable in the serum as early as 1 h post treatment. These cytokines were not produced in mice lacking NKT cells (CD1d-/-), whose exclusive involvement in this biological activity was further confirmed by intracellular immuno-staining. Growth factor production was accompanied by significant changes in the myeloid compartment of treated mice, namely mobilization of myeloid progenitors (colony-forming unit cells, CFU-C) and neutrophils from the bone marrow to the periphery. Taken together, our data support the notion that activated NKT cells influence innate immune responses by recruiting myeloid progenitors and granulocytes to the periphery through their production of hematopoietic growth factors.     

A comparison of in vitro, immuno-suppressive and clinical effects of goat and horse anti-human thymocyte globulin in macaca monkeys

Anti-human thymocyte globulin was raised in four goats and two horses. The in vitro properties were defined, and clinical effects and immunosuppressive efficacy were tested in Macaca monkeys bearing skin xenografts or allografts. All goat and one horse ALG preparation produced significant graft prolongation; one horse ALG showed no in vivo immune suppression. Graft survival correlated well with the rosette-inhibiting activities of the various ALG batches. Goat ALG was well tolerated and produced no local, systemic or anaphylactic reactions; both horse ALG preparations consistently produced local reactions, serum sickness, and, occasionally severe anaphylactic reactions in sensitized and non-sensitized monkeys. Prolonged treatment with goat or horse ALG for up to 10 months caused lymphocyte depletion and plasmacytosis in lymph nodes and spleen but no other apparent pathological changes in a wide variety of tissues studied; infective complications did not occur. Goat IgG was more immunogenic than horse but circulating antibody titres to xenogenic IgG could not be related to clinical responses to ALG, to deposition of xenogenic IgG in kidneys, or to immunosuppressive potency of ALG batches. It was concluded that goat ALG was consistently more potent and less toxic than horse ALG.     

Bone marrow T cell colony-forming cells: studies of their origin and use in monitoring T cell-depleted bone marrow grafts.

Peripheral blood T cell colony forming cells (T-CFC) are mature T cells. When blood was fractionated into sheep red blood cell receptor positive (E+) and negative (E-) fractions, T cell colony growth was largely restricted to the E+ population. When bone marrow was similarly fractionated, many colonies grew from the E- cells but myeloid colonies also grew making interpretation of colony numbers difficult. We have, therefore, also assessed T cell proliferation during culture as an expansion index (EI) by determining the absolute number of T cells pre and post culture. This data shows that T cell expansion is on average nine times greater in the E- marrow fraction than in the E+ fraction. Studies are presented suggesting that this is because marrow E- cells contain appropriate accessory cells and that high numbers of T cells inhibit T cell growth. The cells giving rise to bone marrow T cell colonies thus appear to be contaminating mature T cells rather than pre-thymic progenitor cells. We have measured T cell expansion in culture as a sensitive assay of T cell contamination following procedures to remove T cells from bone marrow grafts for the prevention of graft versus host disease (GVHD).     

The use of colony stimulating factors in clinical bone marrow transplantation.

The use of colony stimulating factors in clinical bone marrow transplantation is rapidly evolving. Both granulocyte colony stimulating factor and granulocyte macrophage colony stimulating factor have shown efficacy in bone marrow transplant patients in accelerating the rate of myeloid recovery. The use of colony stimulating factor primed peripheral blood progenitor cells appears to accelerate platelet recovery as well as myeloid recovery.     

The Polyclonal B Cell Activator Dextran-Sulphate Induces Formation of Colony Stimulating Activity

The effect of dextran-sulphate (DS), a polyclonal activator known to stimulate immature murine B-cells, was assayed in a culture system allowing the growth of myeloid cells. It was known that DS induced the production of a myeloid colony stimulating factor (CSF) by cells from both spleen and bone marrow. Nylonwool purified mouse spleen cells, enriched for T cells, showed a dimished CSF production in response to DS, while CSF production in response to Con A was increased. Furthermore, DS induced CSF in both spleen and bone marrow cells from nude mice. Removal of macrophages did not affect CSF production. The CSF induced was non-dialysable and no small molecular weight or lipoprotein inhibitors could be demonstrated. The results suggest that DS activates cells other than T cells or macrophages (possibly B cells or null cells) to produce a myeloid stem cell stimulating substance. These results indicate that interactions between lymphoid and myeloid cells can take place during differentiation.     

Antigen-induced proliferation in vitro of bone marrow precursors of granulocytes and macrophages

The injection of polymerized flagellin into C57BL mice increased the number of granulocyte cluster-forming cells and macrophage colony-forming cells in the bone marrow. The addition of polymerized flagellin to agar cultures of bone marrow cells from normal or antigen-injected mice increased the number and growth rate of macrophage colonies developing when mouse serum was used as the source of colony stimulating factor (CSF). Polymerized flagellin had no potentiating effect on colony growth when urine or medium conditioned by bone marrow, spleen, lymph node or thymic cells were used as the source of CSF. Antigens may stimulate the proliferation of granulocyte and macrophage precursor cells by an indirect mechanism involving the production by bone marrow cells of CSF, or a similar growth promoting factor.